The goal of our laboratory is to develop methods for the induction of peripheral T cell tolerance and prevention of graft-versus-host disease (GVHD) after human hematopoietic cell transplantation (HCT). Transplantation can cure a variety of benign and malignant hematopoietic disorders, but GVHD remains the primary case of transplant-related morbidity, disability and mortality, thereby limiting the use of HCT. Donor T cells that are included in the stem cell inoculum and recognize recipient alloantigens mediate GVHD. An ideal approach to prevent GVHD is to selectively deplete the alloactivated T cells with a short course of treatment early after transplantation, while sparing other T cells that would be responsible for immune reconstitution and control of pathogens. Depleting alloreactive T cells may tip the balance towards regulation and tolerance, if regulatory T cells (Tregs) are also spared. We have evaluated the effects of TCN on T cell activation and found that TCN suppresses DNA synthesis, proliferation and promotes apoptosis in vitro. Based on these preliminary data and the evidence in literature that AKT promotes T cell activation, survival, Th1 cytokine production but suppresses Treg generation, we reason that treatment of TCN would prevent the development of GVHD after allogeneic BMT. This grant would generate the required preliminary data for testing TCN for GVHD prevention in patients undergoing allogeneic BMT. If the proposed approach is effective in preventing GVHD in humans, more patients would benefit from BMT and achieve cure. PUBLIC HEALTH RELEVANCE: AKT, a phosphatidylinositol 3-kinase (PI3K)-dependent serine-threonine kinase, promotes glucose metabolism and survival of activated T cells and limits differentiation of regulatory T cells (Tregs). While AKT is critical for thymocyte development, some authors have proposed that AKT is not essential for survival of resting T cells. These functions of AKT make it an attractive target in controlling GVHD. The objective of this proposal is to control acute GVHD in pre- clinical models of allogeneic bone marrow transplantation (BMT) by targeting the AKT pathway. A small molecule AKT inhibitor, triciribine (TCN), suppress the phosphorylation of AKT thereby inhibiting its activity. Treatment with TCN potently inhibits growth in nude mice of human cancer cells that over-express AKT. We plan to develop sufficient preclinical data to allow the design of a clinical GVHD prevention trial in humans. Our central hypothesis is that treatment of TCN suppresses T cell activation, expansion and survival while promoting Treg generation in response to alloantigen in vivo, and thus prevents GVHD development after allogeneic BMT. To test this hypothesis, we will pursue the following two Specific Aims: 1) To determine the effects of TCN on GVHD, immune reconstitution and GVL activity after allogeneic BMT. Our preliminary data indicate that TCN suppresses T cell response to alloantigen and promotes apoptosis of antigen-activated T cells in vitro, suggesting that treatment of TCN may prevent GVHD after allogeneic BMT in vivo. In this Specific Aim, we will test the efficacy of TCN in the prevention of GVHD in pre-clinical murine models of BMT. Furthermore, we will evaluate the effects of TCN on marrow reconstitution and GVL activity, the beneficial effects of donor T cells. 2) To define the mechanisms of TCN in the prevention of GVHD. Multiple mechanisms may be involved in the prevention of GVHD by TCN. In this Specific Aim, we will evaluate the potential effects of TCN on T cell expansion, apoptosis, cytokine production, trafficking and Treg generation, which are critical factors in the development of GVHD. To accomplish these objectives, our team has expertise in immunology, bone marrow transplantation and drug therapy. Unique transgenic, knock-out and knock-in mice are available for all the planned experiments. The proposed studies will definitely evaluate the efficacy and mechanisms of TCN in controlling GVHD in clinically relevant models of BMT, and may point to a novel and more selective strategy to prevent GVHD in the clinic.